The present invention relates to a universal immunomodulatory cytokine-expressing bystander cell line, a composition comprising such a cell cancer antigen, a method of making such a cell line, and a method of using such a composition.
Cancer immunotherapy is a therapeutic treatment of cancer. It is based on the premise that the failure of the immune system to reject spontaneously arising tumors is related to the failure of the immune system to respond appropriately to tumor antigens. In a functioning immune system, tumor antigens are processed and expressed on the cell surface in the context of major histocompatibility complex (MHC) class I and II molecules, which, in humans, are also referred to as xe2x80x9chuman leukocyte associatedxe2x80x9d(HLA) molecules. When complexed to antigens, the MHC class I and II molecules are recognized by CD8+ and CD4+ T-cells, respectively. This recognition generates a set of secondary, cellular signals and the paracrine release of specific cytokines or soluble so-called xe2x80x9cbiological response modifiersxe2x80x9d that mediate interactions between cells and stimulate host defenses to fight off disease. The release of cytokines then results in the proliferation of antigen-specific T-cells.
Active immunotherapy involves the injection of cancer or tumor cells to generate either a novel or an enhanced systemic immune response. The tumor cells employed can be autologous, i.e., derived from the host to be treated, or allogeneic, i.e., derived from a host other than the one to be treated. Such a strategy is referred to as a xe2x80x9cvaccine,xe2x80x9d meaning use of an antigen source, such as an intact cancer or tumor cell, to stimulate an immune response against established metastatic cancerxe2x80x94not prophylactic immunization.
The use of autologous tumor cells as xe2x80x9cvaccinesxe2x80x9d to augment anti-tumor immunity has been extensively investigated (Oettgen et al., in Biologic Therapy of Cancer, DeVita et al., eds. (Lippincott, Philadelphia, Pa.), pp. 87-119 (1991)). Although a few patients appear to have benefited from autologous cancer vaccines, their use has only realized partial and short-lived results. Thus, numerous attempts have been made to improve the efficacy of cancer vaccines. Such attempts include radiation and/or chemical modification, infection of autologous tumor cells with virus prior to reinjection into a patient, and transfection/transduction of the tumor cells with genes encoding immunologically relevant molecules, such as cytokines or T-cell co-stimulatory molecules. These attempts, which have been initially explored in murine tumor models, have demonstrated the ability to prime systemic immune responses capable of mediating the rejection of micrometastatic tumors at distant sites. Analysis of the mechanisms of the anti-tumor immune responses generated through such vaccination has underscored the importance of the T-cell arm of the immune system in tumor rejection. Nonspecific immunostimulants also have been used, although little improvement has been realized.
At the clinical level, transfection/transduction of tumor cells with genes encoding immunologically relevant molecules involves tumor resection, culture of cells isolated from the tumor, transfection/transduction of the cultured tumor cells with a gene encoding an immunologically relevant molecule, such as a cytokine, e.g., GM-CSF, irradiation of the transfected/transduced tumor cells, and administration of the irradiated tumor cells to the patient. Tumor cells that have been genetically modified to express various factors, such as IL-4, IL-2, IFN-xcex3, TNF-xcex1, G-CSF, JE, IL-7 and IL-6, have been shown to lead to rejection of the genetically modified cells in syngeneic hosts (Tepper et al., Cell 57: 503-512 (1989); Li et al., Mol. Immunol. 27: 1331-1337 (1990); Golumbek et al., Science 254: 713-176 (1991); Fearon et al., Cell 60: 397-403 (1990); Gansbacher et al., J. Exp. Med. 172: 1217-1224 (1990); Gansbacher et al., Cancer Res. 50: 7820-7825 (1990); Watanabe et al., PNAS USA 86: 9456-9460 (1989); Asher et al., J. Immunol. 146: 3227-3234 (1991); Blankenstein et al., J. Exp. Med. 173: 1047-1052 (1991); Teng et al., PNAS USA 88: 3535-3539 Exp. Med. 173: 889-897 (1991); Rollins et al., Mol. Cell. Biol. 11: 3125-3131 (1991); Hock et al., J. Exp. Med. 174: 1291-1298 (1991); Aoki et al., PNAS USA 89: 3850-3854 (1992); Porgador et al., Cancer Res. 52: 3679-3686 (1992)). Systemic immunity has been demonstrated to increase with cells that express IL-4, IL-2, IFN-xcex3, TNF-xcex1, IL-7 or IL-6 (Golumbek et al. (1991), supra; Porgador et al. (1992), supra).
Various studies comparing irradiated, cytokine-transduced autologous tumor cells have demonstrated that GM-CSF-transduced autologous tumor cells are the most potent inducers of long-lasting, specific tumor immunity (Dranoff et al., PNAS USA 90: 3539-3543 (1993); see, also, Asher et al., J. Immunol. 146: 3327-3334 (1990); Sanda et al., J. of Urology 151: 622-628 (1994); Simons et al., Cancer Research 57: 1537-1546 (1997)). The efficacy of GM-CSF-transduced vaccines has been demonstrated in preclinical models of melanoma, lymphoma, and cancers of the lung, colon, kidney and prostate (Dranoff et al. (1990), supra; Golumbek et al. (1991), supra; Sanda et al. (1994), supra; Jaffee et al., J. Immunother. 18: 1-9 (1995); Caducci et al., Cancer (Phila.) 75: 2013-2020 (1995); Vieweg et al., Cancer Res. 54: 1760-1765 (1994); Jaffee et al., J. Immunother. 19: 1-8 (1996); Levitsky et al., J. Immunol. 156: 3858-3865 (1996)). At the site of vaccination, GM-CSF locally activates (paracrine) antigen-presenting cells (APCs), including dendritic cells and macrophages. APCs subsequently prime CD4+ and CD8+ T-cells, which recognize tumor-associated antigens at metastatic sites, thereby mediating systemic antitumor immunity.
A number of phase-I clinical trials in patients with metastatic cancer have taken place. At Johns Hopkins University, patients with metastatic renal cell carcinoma were treated either with unmodified irradiated autologous tumor cells or irradiated autologous tumor cells transduced to secrete GM-CSF. Measured parameters of immunity paralleled what had been seen in the mouse models and the randomization enabled a clear demonstration of the role of GM-CSF as a molecular adjuvant. A subsequent trial in the treatment of patients with metastatic prostate cancer with autologous GM-CSF-transduced tumor cells extended these observations. Ongoing is a trial at the Dana Farber Cancer Institute in which patients with metastatic melanoma are being treated with autologous GM-CSF-transduced tumor cells.
The pilot studies at Johns Hopkins University and the Dana Farber Cancer Institute and elsewhere have lent support to the use of irradiated, cytokine-transduced autologous tumor vaccines as a therapeutic method of treatment.For many malignancies, large numbers of autologous tumor cells are easily obtained at presentation prior to surgery or chemotherapy-induced remission. For diseases such as acute or chronic leukemias, lymphoma, and colonic carcinoma, well over 5xc3x97109 tumor cells can be obtained and stored with methodologies currently in use at most cancer treatment centers. However, the need for in vitro culture to enable gene transfer and the inability to obtain reproducibly and uniformly high levels of GM-CSF production through such procedures limits this therapeutic approach.
In order to circumvent this problem, a number of investigators are conducting studies of immunization with irradiated, GM-CSF-transfected allogeneic tumor cell lines, such as in the treatment of prostate and pancreatic cancer. The rationale for this approach is that the relevant tumor antigen(s) may be shared between the immunizing allogeneic tumor cell line and the tumor of the patient who is being immunized. Given that the relevant tumor antigens have not been defined in most of these systems, this assumption remains as yet unproven.
In view of the above, materials and methods that would obviate the need for in vitro culture for purposes of gene transfer to autologous tumor cells and that would enable reproducible and uniform immunomodulatory cytokine, e.g., GM-CSF, production would be highly desirable. Therefore, it is an object present invention to provide such materials and methods. This and other objects and advantages will become apparent from the detailed description provided herein.
The present invention provides a universal immunomodulatory cytokine-expressing bystander cell line. The universal bystander cell line is a line, which either naturally lacks major histocompatibility class I (MHC-I) antigens and major histocompatibility class II (MHC-II) antigens or is modified so that it lacks MHC-I antigens and MHC-II antigens. In addition, the universal bystander cell line is modified by introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding an immunomodulatory cytokine operably linked to a promoter. Preferably, the immunomodulatory cytokine is granulocyte macrophage-colony stimulating factor (GM-CSF). The universal bystander cell line expresses preferably at least about 500 ng, more preferably at least about 1,000 ng, GM-CSF /106 cells/24 hours. Alternatively and also preferably, the immunomodulatory cytokine is interleukin 2 (IL-2). Preferably, the human cell line is characterized by the absence of B-lymphocyte markers of immunoglobulin, an Epstein-Barr virus (EBV) genome and an associated nuclear antigen, and receptors for EBV. A preferred human cell line is one that is derived from a blast crisis of chronic myeloid leukemia. An example of a preferred cell line is K562. Preferably, the universal bystander cell line grows in defined, i.e., serum-free, medium. The promoter to which the nucleic acid sequence encoding an immunomodulatory cytokine is operably linked is preferably a cytomegalovirus promoter. Preferably, the universal bystander cell line further comprises a nucleic acid sequence encoding hygromycin resistance operably linked to a promoter and is selected by growth in culture medium comprising at least about 400 xcexcg/ml hygromycin, preferably followed by growth in culture medium comprising at least about 1,000 xcexcg/ml hygromycin.
Also provided by the present invention is a composition comprising the universal bystander cell line and a cancer antigen. A method of making a universal immunomodulatory cytokine-expressing bystander cell line is also provided, as well as a method of stimulating an immune response to a cancer in a human patient.